CD86 and CD80 receptor competition assays

ABSTRACT

The present invention discloses a method for assaying the binding of L104EA29YIg to a receptor. The receptor is preferably CD86 or CD80. The present invention also discloses antibodies to be used in the assay, as well as hybridomas expressing the antibodies.

CROSS REFERENCE TO OTHER APPLICATIONS

This application claims the benefit of priority from provisional U.S.Patent Application 60/761,624, filed Jan. 24, 2006, and provisional U.S.Patent Application 60/832,012, filed Jul. 20, 2006.

FIELD OF THE INVENTION

The present invention relates to methods for monitoring compounds usedto treat immune system diseases such as graft rejection following organtransplant. Specifically, the present invention relates to aflow-cytometry based CD86 or CD80 receptor competition based assay formeasuring the binding efficiency of L104EA29YIg to CD86 or CD80receptors by comparing the binding of non-competing anti-CD86 or CD80monoclonal antibodies to CD86 or CD80 receptors (total CD86 or CD80expression) respectively and the binding of competing anti-CD86 or CD80antibodies to CD86 or CD80 receptors not bound by L104EA29YIg.

BACKGROUND OF THE INVENTION

The hallmark of a vertebrate immune system is the ability todiscriminate “self” from “non-self” (foreign). This property has led tothe evolution of a system requiring multiple signals to achieve optimalimmune activation (Janeway, Cold Spring Harbor Symp. Quant. Biol.54:1-14 (1989)). T cell-B cell interactions are essential to the immuneresponse. Levels of many cohesive molecules found on T cells and B cellsincrease during an immune response (Springer et al., A. Rev. Immunol.5:223-252 (1987); Shaw and Shimuzu, Current Opinion in Immunology, Eds.Kindt and Long, 1:92-97 (1988)); and Hemler Immunology Today 9:109-113(1988)). Increased levels of these molecules may help explain whyactivated B cells are more effective at stimulating antigen-specific Tcell proliferation than are resting B cells (Kaiuchi et al., J. Immunol.131:109-114 (1983); Kreiger et al., J. Immunol. 135:2937-2945 (1985);McKenzie, J. Immunol. 141:2907-2911 (1988); and Hawrylowicz and Unanue,J. Immunol. 141:4083-4088 (1988)).

The generation of a T lymphocyte (“T cell”) immune response is a complexprocess involving cell-cell interactions (Springer et al., A. Rev.Immunol. 5:223-252 (1987)), particularly between T and accessory cellssuch as B cells, and production of soluble immune mediators (cytokinesor lymphokines) (Dinarello and Mier, New Engl. Jour. Med 317:940-945(1987)). This response is regulated by several T-cell surface receptors,including the T-cell receptor complex (Weiss et al., Ann. Rev. Immunol.4:593-619 (1986)) and other “accessory” surface molecules (Springer etal., (1987)supra). Many of these accessory molecules are naturallyoccurring cell surface differentiation (CD) antigens defined by thereactivity of monoclonal antibodies on the surface of cells (McMichael,Ed., Leukocyte Typing III, Oxford Univ. Press, Oxford, N.Y. (1987)).

In order to achieve effective T lymphocyte activation, two receptors onthe cell surface must be engaged by their respective ligands and delivera signal to the cell. First the T cell receptor must recognize antigenin the context of MHC on an antigen presenting cell. Second, aco-stimulatory receptor must bind the appropriate ligand, orco-receptor, on the antigen presenting cell. The most studied T cellco-stimulatory receptor is CD28, which binds to B7 molecules (CD80 andCD86) on antigen presenting cells. Green J L, Leytze G M, Emswiler J,Peach R, Bajorath J, Cosand W, Linsley P S. Covalent dimerization ofCD28/CTLA-4 and oligomerization of CD80/CD86 regulate T cellcostimulatory interactions. J. of Biol. Chem. 271: 26762-26771, 1994.Inhibition of the CD28/B7 pathway in vitro inhibits T cellproliferation, cytokine production and induces antigen specific T cellunresponsiveness. Green J L, Leytze G M, Emswiler J, Peach R, BajorathJ, Cosand W, Linsley P S. Covalent dimerization of CD28/CTLA-4 andoligomerization of CD80/CD86 regulate T cell costimulatory interactions.J. of Biol. Chem. 271: 26762-26771, 1994; and Kelly S, Linsley P, WarnerG, Shyu W C and Paborji M. Investigator Brochure, BMS-188667, CTLA4Ig.In animal models, this pathway has been shown to be important in Tcell-dependent immune responses, including alloantigen recognition andautoimmunity. Green J L, Leytze G M, Emswiler J, Peach R, Bajorath J,Cosand W, Linsley P S. Covalent dimerization of CD28/CTLA-4 andoligomerization of CD80/CD86 regulate T cell costimulatory interactions.J. of Biol. Chem. 271:26762-26771, 1994; and Kelly S, Linsley P, WarnerG, Shyu W C and Paborji M. Investigator Brochure, BMS-188667, CTLA4Ig.Larsen, C. P., Pearson, T. C., Adams, A. B., Tso, P., Shirasugi, N.,Strobert, E., Anderson, D., Cowan, S., Price, K., Naemura, J., Emswiler,J., Greene, J., Turk, L., Bajorath, J., Townsend, R., Hagerty, D.,Linsley, P. S., and R. J. Peach. 2005. Rational Development of LEA29Y, aHigh-Affinity Variant of CTLA4-Ig with Potent ImmunosuppressiveProperties. American Journal of Transplantation. 5(3):443-53. Thus, theCD28/B7 pathway represents a viable, logical target for animmunomodulatory therapeutic agent.

CTLA4Ig (BMS-188667), a fusion protein comprising the extracellulardomain of human CTLA-4 (cytotoxic T-lymphocyte-associated antigen 4) anda fragment of the Fc domain of human IgG1, blocks the CD28/B7 pathway bybinding to CD80 and CD86 on the surface of the antigen presenting cells.This compound has been found to be clinically useful as animmunosuppressant. See U.S. patent application Ser. No. 10/419,008(Publication No. 20040022787 A1), hereby incorporated by reference inits entirety, which describes and discusses CTLA4Ig and L104EA29YIg andmethods of preparation and use thereof. U.S. Pat. Nos. 5,844,095,5,885,796, and 5,851,795, also incorporated by reference in theirentirety, describe and discuss CTLA4Ig.

A related molecule, L104EA29YIg (BMS-224818) (also known as LEA29Y), wasfound to be a particularly potent immunomodulatory therapeutic agent.This compound is a human CTLA4Ig molecule containing a two amino acidsubstitution that results in enhanced binding to CD80 and CD86 relativeto CTLA4Ig. See Larsen, C. P., Pearson, T. C., Adams, A. B., Tso, P.,Shirasugi, N., Strobert, E., Anderson, D., Cowan, S., Price, K.,NaemurFfia, J., Emswiler, J., Greene, J., Turk, L., Bajorath, J.,Townsend, R., Hagerty, D., Linsley, P. S., and R. J. Peach. 2005.Rational Development of LEA29Y, a High-Affinity Variant of CTLA4-Ig withPotent Immunosuppressive Properties. American Journal ofTransplantation. 5(3):443-53. U.S. patent application Ser. No.09/865,321 (Publication No. 2002-0182211 A1), which is also herebyincorporated by reference in its entirety, describes and discussesL104EA29YIg.

CD80 and CD86 are discussed in Carreno, B. M., and Collins, M., 2002(The B7 Family of Ligants and Its Receptors. New Pathways forCostimulation and Inhibition of Immune Responses, Annu. Rev Immunol.20:29-53) and Salomon, B., and Bluestone, J. A., 2001 (Complexities ofCD28/B7: CTLA-4 Costimulatory Pathways in Autoimmunity andTransplantation, Annu. Rev. Immunol. 19:225-52). Given that CTLA4Ig andL104EA29YIg bind to circulating leucocytes expressing CD80 and/or CD86molecules, it would be informative to monitor the extent to which CD80and/or CD86 is bound to the fusion protein(s), in addition to the amountof compound circulating in the plasma during clinical use. In doing so,clinicians would be able to correlate compound exposure levels withreceptor saturation levels required for efficacy in order to monitorbinding efficiency. Understanding the extent to which CD86 is saturatedwith L104EA29YIg at various blood concentrations can be used to helpjustify different dosing schemes or regimes. For example, during thedevelopment phase, different formulations and routes of administrationwill be utilized (e.g. monthly intravenous or weekly subcutaneoustreatment). This assay could be used to help establish the best routeand course of administration which demonstrates maximum saturation forthe longest period of time.

BRIEF SUMMARY OF THE INVENTION

The present invention relates to assays for monitoring and measuring thebinding of L104EA29YIg to a receptor, in particular, a CD86 or CD80receptor. In this assay, peripheral mononuclear cells are isolated froma blood sample, and portions of the mononuclear cell sample arepreincubated with increasing concentrations of L104EA29YIg. Afterincubation, a labeled anti-CD86 or anti-CD80 antibody is added, and thebinding of the labeled antibody is measured using flow cytometry. Bycomparing the binding of portions of the mononuclear cell sample withvarying concentrations of L104EA29YIg added to a portion of themononuclear cell sample with no L104EA29YIg added, one can determinethat L104EA29YIg is binding the receptor.

In yet another embodiment of the invention, an assay method is providedin which the binding efficiency of L104EA29YIg to a receptor is measuredusing a competing antibody and a non-competing antibody concurrently. Insuch an assay, peripheral monocytes are isolated from a blood sample,and portions of the monocyte sample are preincubated with L104EA29YIg.After incubation, a labeled competing anti-receptor antibody is added,and then a non-competing anti-receptor antibody is added. The binding ofthe two antibodies is measured using flow cytometry. The binding of thenon-competing antibody represents the amount of total receptor, and thebinding of the competing antibody represents the amount of availablereceptor, unbound by L104EA29YIg. In this way, the binding efficiency ofL104EA29YIg may be determined using a single sample.

In yet another embodiment of the invention, an assay method is providedin which the binding efficiency of L104EA29YIg to a receptor is measuredusing a competing antibody and a non-competing antibody in separatesamples. In such an assay, whole blood is treated with L104EA29YIg. PBSis used as an untreated control. Mouse IgG solution is added to allsamples to block non-specific binding of detection reagents. To detectlevels of unbound CD86, fluorescently labeled competing anti-receptorantibody (e.g., mAb HA5) is added to one set of samples. To detect totalCD86 levels, a fluorescently labeled non-competing anti-receptorantibody (e.g. mAb 2D4) is added to another set of samples. To detectmonocytes, CD14-FITC is added to each sample. To assess non-specificfluorescence associated with the labeled anti-CD86 mAbs, excessunlabeled anti-human CD86 mAb is added to a subset of the relevantsamples (e.g. unlabeled HA5 is added to samples containing labeled HA5).Cells are lysed using Lysing solution, and the binding of the antibodiesis measured using flow cytometry. The binding of the non-competingantibody represents the amount of total receptor, and the binding of thecompeting antibody represents the amount of available receptor, unboundby L104EA29YIg. Specific binding (ΔMFI (Medium fluorescence intensity))is determined by the difference between the total binding (labeledanti-CD86 mAb alone) and the non-specific binding (labeled + excessunlabeled anti-CD86 mAb). In this way, the binding efficiency ofL104EA29YIg may be determined using two separate samples.

In yet another embodiment of the invention, an assay method is providedfor monitoring the binding efficiency of L104EA29YIg in a clinicalsetting. In such an assay, a patient is dosed with L104EA29YIg. A bloodsample from the patient is obtained, and a mixture of mouse IgGs areadded to aliquots of the blood sample to reduce Fc receptor-mediatednon-specific binding. Human CD14-FITC is added to identify monocytes.Then, labeled competing anti-receptor antibody and labeled non-competinganti-receptor antibody are added to the aliquots of blood sample. Asabove, to assess non-specific fluorescence associated with the labeledanti-CD86 mAbs, excess unlabeled anti-human CD86 mAb is added to asubset of the relevent samples (e.g. unlabeled HA5 is added to samplescontaining labeled HA5). A lysing/fixative solution is used to lyse redblood cells and fix leukocytes. The samples are centrifuged to removelysed blood cells and isolate leukocytes. The total receptor andavailable receptor (not bound by L104EA29YIg) are measured bydetermining the binding of the competing and non-competing antibodies(respectively). Specific binding (ΔMFI) is determined by the differencebetween the total binding (labeled anti-CD86 mAb alone) and thenon-specific binding (labeled+excess unlabeled anti-CD86 mAb).

In one preferred aspect of the above embodiments of the presentinvention, the receptor is CD86 or CD80.

In another preferred aspect of the above embodiments, the anti-receptorantibody is an anti-CD86 antibody. In particular, the competinganti-CD86 antibody is FUN-1, IT2.2, or HA5 (clone HA5.2B7). In anotherpreferred embodiment, the anti-receptor antibody is an anti-CD80antibody. In particular, the competing anti-CD80 antibody is either mAbL307.4 or mAb MAB104.

In another preferred aspect of the invention, the anti-human CD86 oranti-human CD80 antibody is labeled with a fluorophore. In anotherpreferred aspect of the invention, the fluorophore is phycoerythrin(PE).

In yet another preferred aspect of the above embodiments, thenon-competing anti-CD86 antibody is mAb 2D4.

In yet another preferred aspect of the above embodiments, thenon-competing anti-CD80 antibody is mAb 1420.

In yet another embodiment of the invention, monoclonal antibodies mAb1420 and 2D4, and hybridomas that express such mAbs are provided. Thehybridomas used to produce these antibodies were deposited at the ATCCon Jan. 13, 2006, as deposit numbers PAT-7304 (hybridoma expressingmAb1420), and PAT-7305 (hybridoma expressing mAb 2D4). These and otherembodiments of the invention will be apparent in light of the detaileddescription below.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 illustrates the results of a CD86 competition in isolatedperipheral blood mononuclear cells with competing mAb FUN-1.

FIG. 2 illustrates (a) the results of competition in whole blood withcompeting mAb FUN-1 (1 ug/ml) and (b) the results of competition inwhole blood with a non-competing mAb 2D4.

FIG. 3 presents the nucleotide sequence (SEQ ID NO:1) of the CTLA4Igmolecule. Also shown is the amino acid sequence (SEQ ID NO:2) encoded bythe nucleic acid. CTLA4Ig molecules that can be produced from thisnucleotide sequence include molecules having the amino acid sequence ofresidues: (i) 26-383 of SEQ ID NO:2, (ii) 26-382 of SEQ ID NO:2, (iii)27-383 of SEQ ID NO:2,or (iv) 26-382 of SEQ ID NO:2, or optionally (v)25-382 of SEQ ID NO:2, or (vi) 25-383 of SEQ ID NO:2. The DNA and aminoacid sequences comprise the following regions: (a) an Oncostatin Msignal sequence (amino acids 1-26 of SEQ ID NO:2); (b) an extracellulardomain of human CTLA4 (amino acids 27-151 of SEQ ID NO:2); (c) amodified portion of the human IgG1 constant region (amino acids 152-383of SEQ ID NO:2), including a modified hinge region (amino acids 152-166of SEQ ID NO:2), a modified human IgG1 CH2 domain (amino acids 167-276of SEQ ID NO:2), and a human IgG1 CH3 domain (amino acids 277-383 of SEQID NO:2).

FIG. 4 depicts a nucleotide (SEQ ID NO:3) and amino acid (SEQ ID NO:4)sequence of L104EA29YIg (also known as “LEA29Y”) comprising anOncostatin M signal sequence; a mutated extracellular domain of CTLA4starting at (as designated in FIG. 4) methionine at position +1 andending at aspartic acid at position +124, or starting at alanine atposition −1 and ending at aspartic acid at position +124; and an Igregion. SEQ ID NO: 3 and 4 designate the first amino acid of theOncostatin M signal sequence (M, which is followed by G) as 1. SEQ IDNO: 3 and 4 depict a nucleotide and amino acid sequence, respectively,of L104EA29YIg comprising an Oncostatin M signal sequence; a mutatedextracellular domain of CTLA4 starting at methionine at position +27 andending at aspartic acid at position +150, or starting at alanine atposition +26 and ending at aspartic acid at position +150; and an Igregion. L104EA29YIg can have the amino acid sequence of residues: (i)26-383 of SEQ ID NO:4, (ii) 26-382 of SEQ ID NO:4; (iii) 27-383 of SEQID NO:4 or (iv) 27-382 of SEQ ID NO:4, or optionally (v), 25-382 of SEQID NO:4, or (vi) 25-383 of SEQ ID NO:4.

FIG. 5 depicts the results of an ex vivo CD86 receptor competition inwhole blood with mAb HA5 and mAb FUN1. (NHV=Normal healthy volunteer.)This figure demonstrates that the concentration of L104EA29YIg requiredto inhibit specific binding of HA5 by 50% is 0.13 ug/ml, and theconcentration of L104EA29YIg required to inhibit the specific binding ofFUN1 by 50% is 0.49 ug/ml.

FIG. 6 depicts the characterization of CD86 competition with mAb FUN1after incubation of whole blood with L104EA29YIg. (MFI=Medianfluorescence intensity; MLR=Mixed leukocyte reaction; NS=Non-specific).This figure summarizes FUN-1 performance in the assay on blood collectedfrom 6 different NHVs demonstrating the effect of L104EA29YIg inhibitionof antibody binding.

FIG. 7 depicts the characterization of CD86 competition with mAb HA5after incubation of whole blood with L104EA29YIg. This figure summarizesHA5 performance in the assay on blood collected from 6 different NHVsdemonstrating the effect of L104EA29YIg inhibition of antibody binding.

FIG. 8 depicts a CD86 receptor competition assay on whole bloodcollected from NHVs administered L104EA29YIg subcutaneously.(SC=Sub-cutaneous; squares indicate pre-dose, triangles indicate Day 5,circles indicate Day 14, and diamonds indicate Day 42). FIG. 8demonstrates that L104EA29YIg administration SC to normal healthyvolunteers, inhibits the binding of FUN1 to monocytes by day 5, but thiseffect is reversed by day 14.

FIG. 9 depicts the correlation between pharmacokinetics (PK) andpharmacodynamics (PD) of the results shown in FIG. 8, which helps oneunderstand what serum drug concentrations are required to achieve adesired pharmacodynamic activity such as saturating the target. Open andclosed triangles represent the two subjects who received L104EA29YIg.This figure demonstrates that in the subjects administered L104EA29YIgSC, more inhibition of Fun-1 binding to peripheral blood monocytes isobserved with increasing serum concentrations of L104EA29YIg.

FIG. 10 depicts a comparison of the specific binding of severalanti-CD86 mAbs in NHVs to monocytes in whole blood. The data show thatall three antibodies bind to monocytes in a similar fashion.

FIG. 11 depicts specific binding of mAb 2D4 in clinical samples(including blood from renal transplant patients treated withL104E29YIg). In transplant patients receiving L104EA29YIg, 2D4 bindingis unchanged following administration of L104EA29YIg and notsignificantly different from NHVs.

FIG. 12 depicts specific binding of mAb HA5 and mAb FUN1 in clinicalsamples (including blood from renal transplant patients treated withL104E29YIg). This data demonstrates that as opposed to 2D4, FUN-1 andHA5 binding are significantly inhibited by L104E29YIg administration intransplant patients and significantly reduced compared to NHVs.

FIG. 13 depicts simulated clinical PK and PD profiles. This figuredepicts a model suggesting how this assay could be used to monitorreceptor saturation following L104E29YIg administration. It hypothesizesthat shortly after the first dose, receptor saturation is maximized andremains at the desired level despite changes in dose regimen such asfrequency of dose or strength of dose.

FIG. 14 depicts the titration of anti-CD86 PE FUN1, demonstrating theeffects on L104EA29YIg binding. These results were used to determine theconcentration of antibodies to use in competition assays.

FIG. 15 depicts the titration of anti-CD86 PE HA5.2B7, demonstrating theeffects on L104EA29YIg binding. These results were used to determine theconcentration of antibodies to use in competition assays.

FIG. 16 depicts the titration of anti-CD86 PE 2D4 , demonstrating theeffects of L104EA29YIg binding. L104EA29YIg does not affect the bindingof mAb 2D4.

FIG. 17 depicts specific binding of a non-competing mAb 2D4 in clinicalsamples of long term and short term subjects. In transplant patientreceiving L104EA29YIg, 2D4 binding is unchanged following administrationof L104EA29YIg and not significantly different from Cyclosporine A (CsA)treated control subjects. The assay is similar to the assay described inFIG. 11 except that CsA treated subjects are used as controls andresults from subjects treated with L104EA29YIg for a period of 6 monthsor less are also included.

FIG. 18 depicts specific binding of a competing monoclonal antibody HA5in clinical samples of long term and short term subjects. This datademonstrate that HA5 binding is significantly inhibited by L104EA29YIgadministration in transplant patients and significantly reduced comparedto CsA treated control subjects. The assay is similar to the assaydescribed in FIG. 12 except that CsA treated subjects were used ascontrols and results from subjects treated with L104EA29YIg for a periodof 6 months or less are also included.

FIG. 19 depicts CD86 receptor competition assay in whole blood fromnormal healthy volunteers with mAb HA5. The data demonstrates that theconcentration of CTLA4Ig required to inhibit specific binding of HA5 by50% is 2.63 μg/ml, whereas the concentration of L104EA29YIg required toinhibit specific binding of HA5 by 50% is 0.11 μg/ml. The assay is thesame type of assay as shown in FIG. 5, donor B, except that the resultsin FIG. 19 represent the average response of 6 donors.

FIG. 20 depicts CD80 receptor competition assay in whole blood fromnormal healthy volunteers with mAb L307.4. The data demonstrate that theconcentration of CTLA4Ig required to inhibit specific binding of L307.4by 50% is 0.01 μg/ml, and the concentration of L104EA29YIg required toinhibit specific binding of L307.4 by 50% is 0.01 μg/ml. The resultsrepresent the average response of 6 donors.

FIG. 21 depicts the results of CD80 and CD86 receptor saturation andinhibition of allo-response assay for comparison. Depicted are the IC50sof CTLA4Ig in a mixed leukocyte reaction, in a CD86 receptor competitionassay using mAb HA5, and in a CD80 receptor competition assay using mAbL307.4. Also depicted are the IC50s of L104EA29YIg in a mixed leukocytereaction, in a CD86 receptor competition assay, and in a CD80 receptorcompetition assay.

DETAILED DESCRIPTION OF THE INVENTION

The present invention may be understood more readily by reference to thefollowing detailed description of the preferred embodiments of theinvention and the Examples included herein. This invention describes thedevelopment of a flow cytometry-based “CD86 Receptor Competition Assay”for L104EA29YIg.

Preliminary attempts to develop a CTLA4Ig-based CD80 and/or CD86receptor competition assay failed. Most peripheral blood monocytesexpress very low (or no) levels of CD80 on their surface prior tostimulation, thus developing a CD80 receptor competition assay would behighly challenging. Additionally, the relatively high affinity ofL104EA29YIg for CD86 (relative to CTLA4Ig or mAbs) allows L104EA29YIg tocompete for binding with the anti-CD86 mAbs tested.

Flow cytometry has been used with increasing regularity in clinicallaboratories for immunophenotyping of leukocyte antigens. The advantagesof flow cytometry include speed, sensitivity, precision and objectivity.The components and operation of flow cytometers is well known to thoseskilled in the art and will not be described in detail herein. Forpurposes of a description of such, applicants refer to U.S. Pat. No.5,567,627, issued Oct. 22, 1996 which is incorporated herein byreference in its entirety. It is sufficient to indicate that thecomponents and methodology of flow cytometry can be used to providespecific information on a number of parameters of a sample. For exampleit is possible to provide information on components of different sizeswithin a sample, while simultaneously providing information on signalsof different wavelengths received from different components receivedfrom the sample. Thus, when a sample includes components of varyingsizes and also includes components with labels which emit differentwavelengths of light the flow cytometry data obtained can providemultidimensional information to the user. The present invention utilizesthis technology by providing different types of labeled antibodies andlabeled and unlabelled cells expressing known antigens. By exposing asample to such and thereafter analyzing such within a flow cytometer itis possible to obtain substantial amounts of information regarding theblood in a quick and efficient manner.

In this flow cytometric assay, the total CD86 expression levels onperipheral monocytes are detected by a truly non-competing mAb (e.g.2D4) and the level of CD86 molecules not bound by L104EA29YIg isdetected by a competing anti-CD86 mAb (e.g. FUN-1 and IT2.2).

As used herein, a competing anti-receptor antibody is an antibody whichis measurably prevented from binding to a receptor by a given molecule,such as L104EA29YIg. A non-competing anti-receptor antibody is anantibody which does not measurably prevent binding of a given molecule,such as L104EA29YIg, to the receptor.

As used herein, “CTLA4Ig” or “CTLA4-Ig” refers to a protein moleculehaving the amino acid sequence of residues: (i) 26-383 of SEQ ID NO:2,(ii) 26-382 of SEQ ID NO:2; (iii) 27-383 of SEQ ID NO:2, or (iv) 27-382of SEQ ID NO:2, or optionally (v) 25-382 of SEQ ID NO:2, or (vi) 25-383of SEQ ID NO:2. Expression of CTLA4Ig in mammalian cells can result inthe production of N- and C- terminal variants. CTLA4Ig also refers tomultimeric forms of the polypeptide, such as dimers, tetramers, andhexamers. Dimer combinations can include, for example: (i) and (i); (i)and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii)and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi);(iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and(iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and, (vi)and (vi). These different dimer combinations can also associate witheach other to form tetramer CTLA4Ig molecules. (DNA encoding CTLA4Ig asshown in SEQ ID NO:2 was deposited on May 31, 1991 with the AmericanType Culture Collection (ATCC), 10801 University Blvd., Manassas, Va.20110-2209 under the provisions of the Budapest Treaty, and has beenaccorded ATCC accession number ATCC 68629; a Chinese Hamster Ovary (CHO)cell line expressing CTLA4Ig as shown in SEQ ID NO:2 was deposited onMay 31, 1991 with ATCC identification number CRL-10762).

L104EA29YIg (also known as “LEA29Y” or “L104EA29Y”) is a geneticallyengineered fusion protein similar in structure to CTAL4Ig. Two aminoacid modifications were made to CTLA4Ig, leucine to glutamic acid atposition 104 (L104E), which is position 130 of SEQ ID NO:2, and alanineto tyrosine at position 29 (A29Y), which is position 55 of SEQ ID NO:2,to generate L104EA29YIg.

As used herein, “L104EA29YIg” refers to a protein molecule having theamino acid sequence of residues: (i) 26-383 of SEQ ID NO:4, (ii) 26-382of SEQ ID NO:4; (iii) 27-383 of SEQ ID NO:4, or (iv) 27-382 of SEQ IDNO:4, or optionally (v) 25-382 of SEQ ID NO:4, or (vi) 25-383 of SEQ IDNO:4. Expression of L104EA29YIg in mammalian cells can result in theproduction of N- and C- terminal variants. L104EA29YIg also refers tomultimeric forms of the polypeptide, such as dimers, tetramers, andhexamers. Dimer combinations can include, for example: (i) and (i); (i)and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii)and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi);(iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and(iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and, (vi)and (vi). These different dimer combinations can also associate witheach other to form tetramer L104EA29YIg molecules. (DNA encodingL104EA29YIg was deposited on Jun. 20, 2000, with the American TypeCulture Collection (ATCC) under the provisions of the Budapest Treaty.It has been accorded ATCC accession number PTA-2104. L104EA29YIg isfurther described in co-pending U.S. patent application Ser. Nos.09/579,927, 60/287,576 and 60/214,065, and in US20020182211A1 andWO/01/923337 A2, which are incorporated by reference herein in theirentireties.)

EXAMPLES Example 1 In Vitro Assays

Initial experiments were performed to measure the binding of L104EA29YIgto CD86. Various antibodies were used singly or in combination todevelop an assay procedure that allows the measurement of L104EA29YIgbinding relative to the availability of CD86 receptor.

Materials

-   Dulbecco's PBS without Ca++ and Mg++ (Mediatech Inc., Herndon Va.,    Cat #21-031-CM), or equivalent-   BMS-224818-01 (L104EA29YIg), 100 mg, resuspended and stored frozen    as a 10 mg/ml stock solution in PBS-   Lymphocyte Separation Medium (Mediatech Inc., Herndon Va., Cat    #25-072-CV)-   Sodium azide (Sigma-Aldrich, Milwaukee, Wis., Cat #S2002), or    equivalent-   Trypan Blue, 0.4% (Invitrogen, Grand island N.Y., Cat #15250-061) or    equivalent-   10×FACS Lysing Solution (BD Biosciences, San Jose Calif., Cat    #349202)-   Fetal Bovine Serum (Mediatech Inc., Herndon Va., Cat #35-011-CV), or    equivalent-   Human IgG1κ, from human plasma (Sigma-Aldrich, St. Louis, Mo., Cat    #15154), or equivalent-   IgG from human serum (Sigma-Aldrich, St. Louis, Mo., Cat #14506), or    equivalent-   IgG from murine serum (Sigma-Aldrich, St. Louis, Mo., Cat #15381),    or equivalent-   Anti-CD14-FITC conjugated mAb (Becton Dickinson, San Jose, Calif.    Cat #555397)-   Anti-CD86-PE conjugated mAb, clone FUN-1 (Becton Dickinson, San    Jose, Calif. Cat #555658)-   Anti-CD86-PE conjugated mAb, clone IT2.2 (Becton Dickinson, San    Jose, Calif. Cat #555665)-   Anti-CD86-PE conjugated mAb 2D4-PE. (BMS proprietary mAb, conjugated    to PE, Batch Control #14640, by Caltag Laboratories)-   Anti-CD80-PE conjugated mAb (1420-PE) (BMS proprietary mAb,    conjugated to PE, Batch Control #17312, by Caltag Laboratories.)-   Sodium citrate CPT Vacutainer™, 8 mL, (Becton Dickinson, Cat    #362761)-   Sodium heparin CPT Vacutainer™, 8 mL, (Becton Dickinson, Cat    #362753)-   Anti-CD80 conjugated mAb L307.4 (Becton Dickinson, Cat 340294)-   Anti-CD80 conjugated mAB 104 (Beckman Coulter, Cat #IM1976)-   Anticoagulant Acid Citrate Dextrose Solution Formula (ACD-A) (2    mg/ml dextrose, 1.8 mg/ml sodium citrate, 0.6 mg/ml citric acid;    Gambro #777967000)    Heparin (Sigma H3149, 30 U/ml)    A. Detection of CD86 in Human PBMC and Competition with L104EA29YIg

Given that monocytes represent the circulating leukocyte populationexpressing the most abundant expression of CD86, the expression of CD86on CD14+ monocytes was evaluated in a two color directimmunofluorescence assay (FIG. 1). As expected, peripheral monocytes inPBMCs (peripheral blood monocytes) expressed moderate levels of CD86 asdetected by anti-CD86 mAb FUN-1 (or IT2.2 data not shown) binding. WhenPBMCs are pre-incubated with increasing concentrations of L104EA29YIg(0.1 to 100 μg/test), the detectable anti-CD86 binding was inhibited ina concentration dependant manner. These data suggest that L104EA29YIgcan compete with selective anti-CD86 mAbs (e.g. FUN-1 and IT2.2) forCD86 binding on peripheral blood monocytes. Thus the reagents andpotential for generating a CD86 receptor competition assay to measureco-stimulation blockade by L104EA29YIg exists and does so only due tothe unique high affinity L104EA29YIg has for CD86.

Not wishing to be bound by any specific procedure, the Applicantsprovide the following example procedure to demonstrate how the assay maybe done in an in vitro setting:

PBMC Procedure:

-   -   1. 40 ml blood is drawn into a syringe containing heparin (30        U/ml), EDTA (5.4 mM) or ACD-A (2 mg/ml dextrose, 1.8 mg/ml        sodium citrate, 0.6 mg/ml citric acid) as an anti-coagulant.    -   2. Layer 20 ml of blood over 15 ml of Lymphocyte Separation        Medium.    -   3. Centrifuge 1800 rpm for 25 minutes at room temperature.    -   4. Remove the PBMC layer and transfer to a 50 ml tube. Add 30 ml        PBS.    -   5. Centrifuge 1800 rpm for 10 minutes at room temperature.    -   6. Resuspend the pellet in 50 ml PBS.    -   7. Centrifuge at 1200 rpm for 10 minutes at room temperature.    -   8. Resuspend the pellet in PBS and determine cell number using        trypan blue staining.    -   9. Resuspend the cells at a final concentration of 107 cells per        ml in 0.5% FBS/PBS/0.1% sodium azide.    -   10. Aliquot 106 cells (100 μl) into a 12×75 mm polystyrene tube        on ice.    -   11. Add L104EA29YIg at desired concentrations (e.g., between 0        and 200μg/ml) to tube and incubate for 15 minutes on ice.    -   12. Add 20 μg of either human IgG1 (200 μg/ml final        concentration) or mixed human IgGs (200 μg/ml final        concentration) to block potential FcR binding of detection        antibodies. Incubate 10 minutes on ice.    -   13. Add indicated amount (e.g. 1 μg) of anti-human CD86 PE        (antibody labeled with PE, i.e., phycoerythrin) (e.g. BD clone        FUN1 ) antibody, incubate 30 minutes on ice    -   14. Add 1 ml FACS Lysing Solution, incubate 30 minutes on ice.    -   15. Centrifuge at 1500 rpm for 5 minutes at 4 C. Resuspend        pellet in 250 ul FACS Lysing Solution.    -   16. Read on flow cytometer. For acquisition, gate on the        monocyte population, as identified by forward and side scatter        properties (G1). Acquisition is stopped after accumulation of        5000 G1 events.    -   17. For analysis, the median fluorescence of G1 events is        determined on a histogram of FL2 events (CD86 PE) and is used to        determine the relative level of CD86 present on the surface of        monocytes.        B. Detection of CD86 in Human Whole Blood Samples and        Competition with L104EA29YIg

To be useful as a clinical assay, the CD86 competition observed onPBMC's must also be detectable in whole blood samples. To demonstratethis potential, whole blood from a normal healthy volunteer was drawninto vacutainers containing either ACD or EDTA. The blood was thenpre-incubated with 0, 5, 120 and 250 μg/mL L104EA29YIg at 37° C. for 1hour. Following incubation, CD86 not occupied by L104EA29YIg wasmeasured by incubating the blood samples at 4° C. with FUN-1 (and/orIT2.2). The results of the flow cytometry measurements suggest thatexpected levels of CD86 expression on CD14+ monocytes were detected;however, the use of different anti-coagulants resulted in differentlevels of detectable CD86 in the absence of L104EA29YIg. Increasingconcentrations of L104EA29YIg inhibited FUN-1 binding in a concentrationdependant manner (FIG. 2 a) and there appeared to be no effect ofanticoagulant choice on this inhibition.

Not wishing to be bound by any specific procedure, the Applicantsprovide the following example procedure to demonstrate how the assay maybe done:

Whole Blood Procedure

-   -   1. Draw blood into ACD-A anticoagulant    -   2. Dispense L104EA29YIg into 12×75 mm polypropylene tube for        desired final concentration (e.g., between 0 and 200 μg/ml).    -   3. Add whole blood to L104EA29YIg. Incubate on a rotator in the        37° C. CO₂ incubator for 1 hr.    -   4. Aliquot 200 ul of blood sample into a 12×75 mm polystyrene        tube on ice.    -   5. To each tube, add 25 ug of mouse mixed IgGs to block        potential FcR binding of detection antibodies. Incubate for 15        minutes on ice.    -   6. To each tube, add 20 ul anti-human CD14 FITC (5 μg/ml)/ and        indicated amount (e.g. 1 ug) of anti-human CD86 PE (e.g. BD        clone FUN1). Incubate on ice for 30 minutes.    -   7. Add 2 ml of FACS Lysing Solution (BD) to each tube. Incubate        on ice for 30 minutes.    -   8. Spin the tubes for 5 minutes at 1500 rpm, at 4° C.    -   9. Resuspend in 200 μl FACS Lysing Solution.    -   10. Read on flow cytometer, adjusting compensation settings as        necessary. For acquisition, gate on the monocyte population, as        identified by forward and side scatter properties (G1). Gate on        CD14+ monocytes using a dot plot of forward scatter vs. CD14        (G2). The additive events of G1 and G2 (termed G3) are observed        on a histogram looking at the FL2 channel (CD86 PE). Acquisition        is stopped after accumulation of 3000 G3 events.    -   11. For analysis, the median fluorescence of G3 events is        determined on a histogram of FL2 events (CD86 PE) and is used to        determine the relative level of CD86 present on the surface of        CD14+ monocytes.        C. Detection of CD86 in Human Whole Blood Cells with Concurrent        use of Competing and Non-Competing Anti-CD86 Antibodies.

Additionally, the binding of 2D4, an anti-CD86 mAb which does notcompete with CTLA-4, is not affected by the preincubation with a highconcentration (200 μg/ml) of L104EA29YIg (FIG. 2 b). This suggests that2D4 does not compete with L104EA29YIg for binding to CD86 and can beused to measure the total surface expression of CD86 even in thepresence of L104EA29YIg.

D. Summary

The data for these experiments are shown in FIGS. 1, 2 a and 2 b. Thesedata demonstrate that selected anti-CD86 mAbs (e.g. FUN-1 and IT2.2),which do not compete with CTLA4Ig, will compete with L104EA29YIg forbinding to CD86 on the surface of antigen presenting cells. The dataalso demonstrate that truely non competing anti-CD86 mAbs (e.g. 2D4) canbe used to measure total surface CD86 expression even in the presence ofhigh concentrations of L104EA29YIg. These observations are unique toL104EA29YIg due to the enhanced affinity for CD86 as compared to CTLA4Igand other CD86 ligands. These observations are the basis for the novelapplication of these reagents for the development of a clinical assay tomeasure costimulation blockade by L104EA29YIg.

Example 2 Clinical Ex Vivo and In Vivo Studies

To be useful as a clinical assay, the CD86 competition observed onPBMC's must also be detectable in whole blood samples. To demonstratethis potential, whole blood from a normal healthy volunteer was drawninto vacutainers containing either ACD or EDTA. The blood was thenpre-incubated with varying concentrations of L104EA29YIg. Assayconcentrations were determined by titration of FUN-1 PE, HA5 PE and 2D4PE. (See FIGS. 14-16, and Tables 1 and 2). Following incubation, CD86not occupied by L104EA29YIg was measured by incubating the blood samplesat 4° C. with HA5.2B7. The concentration of L104EA29YIg required toinhibit antibody binding by 50% varies, depending on the antibody. Forexample, 0.13 μg/ml of L104EA29YIg is required to inhibit HA5 binding by50%, while 0.49 μg/ml of L104EA29YIg is required to inhibit FUN-1binding by 50%. (See FIG. 5). FUN-1 and HA5 performance in the assay onblood collected from 6 different normal healthy volunteers (NHVs)demonstrating the effect of L104EA29YIg inhibition of antibody bindingis shown in FIGS. 6 and 7. In general, HA5 appears to be more sensitiveas it detects greater receptor saturation at similar L104EA29YIgconcentrations and lower IC50s. All three monoclonal antibodies (FUN-1,HA5, and 2D4) bind to CD86 (on monocytes) at similar levels. (See FIG.10).

In clinical studies, FUN-1 specific binding, as measured by change inmedian fluorescence intensity (MFI), is inhibited substantially by Day 5after dosing with L104EA29YIg. (See FIG. 8). L104EA29YIg inhibitsbinding of FUN-1, and, when administered subcutaneously, as theconcentration of L104EA29YIg increases, so does the inhibition of FUN-1binding. (See FIG. 9). In transplant patients continually receivingL104EA29YIg, 2D4 binding is unchanged following a single administrationof L104EA29YIg and not significantly different from NHVs. (See FIG. 11).The term “trough” refers to the time point just prior to the nextadministration of the drug, when the concentration of drug is at itslowest blood level in a patient. As opposed to 2D4, Fun-1 and HA5binding are significantly inhibited by L104EA29YIg administration intransplant patients and significantly reduced compared to NHVs. (SeeFIG. 12; 2D4 data not shown.) FIG. 13 is a model suggesting how thisassay could be used to monitor receptor saturation following L104EA29YIgadministration. It hypothesizes that shortly after the first dose,receptor saturation is maximized and remains at the desired leveldespite changes in dose regimen such as frequency of dose or strength ofdose.

Not wishing to be bound by any specific procedure, the Applicantsprovide the following example procedure to demonstrate how the assay maybe done:

Materials

-   L104EA29YIg, BMS Syracuse, lot 4E82288/MSF521A; 100 mg vial    reconstituted with 10 ml PBS, to a final concentration of 10 mg/ml.    50 μl aliquots stored @-20° C.-   CD86 PE (clone HA5.2B7) Beckman Coulter #IM2729 lot 15 (conc 6.25    μg/ml)-   CD86 APC clone 2D4; 0.09 mg/ml; BMS generated mAb, APC conjugated by    Calbiochem-   CD14 FITC BD #555397-   Purified anti-CD86 (clone HA5.2B7) Beckman Coulter #IM2728, 200 μg    lyophilized; resuspended at 200 μg/ml in dH₂O/0.1% azide-   Purified anti-human CD86 (clone 2D4) 2.29 mg/ml-   Mouse IgG, reagent grade, Sigma #15381; 10 mg lyophilized; resuspend    in 5 ml PBS to a final concentration of 2 mg/ml, stored @4° C.-   FACS Lysing Solution, 10×, BD #349202    Whole Blood In Vitro Procedure    -   1. Draw blood into ACD-A vacutainer or syringe with ACD-A as        anti-coagulant.

2. Dilute 10 mg/ml L104EA29YIg in PBS to a concentration that is 10-foldabove the desired final concentration. See dilutions in Table 1 below.TABLE 1 Concentration of L104EA29YIg used in Assays CorrelationL104EA29YIg μl 10 mg/ml with clinical final conc. μl PBS L104EA29YIgvalues 250 μg/ml 150 50 peak 125 μg/ml 175 25 intermediate  5 μg/ml 100First dilute 1:100, trough then add 100  2 μg/ml 160 First dilute 1:100,trough then add 40

-   -   3. Dispense 150 μl of diluted L104EA29YIg into a 12×75 mm        polypropylene tube. Dispense 150 μl PBS to additional tube for        untreated control.    -   4. Add 1.35 ml of whole blood to L104EA29YIg or PBS. Incubate on        a rotator in the 37° C. CO₂ incubator for 1 hr.    -   5. On ice, dispense 10 μl of mouse IgG solution to 12×75 mm        polystyrene sample tubes.    -   6. Dispense 100 μl of blood for each sample into 12-12×75        polystyrene tubes.    -   7. Incubate on ice 10 minutes.    -   8. To three tubes, add 3.5 μl purified, unlabeled HA5 mAb        (purified). To three additional tubes, add 4.4 μl of purified,        unlabeled 2D4 mAb. (See table below). Incubate 15 minutes on        ice.    -   9. To all tubes, add 10 μl CD14 FITC.

10. Add 20 μl of anti-human CD86 PE (clone HA5.2B7) antibody to tubes asindicated in table below. Add 10 μl of 2D4-APC to tubes as indicated inTable 2 below. TABLE 2 Addition of unlabeled/labeled antibody reagents.HAS PE, Unlabeled 2D4- Unlabeled CD14- TUBE μl HAS, μl APC, μl 2D4, μlFITC, μl 1 20 — — — 10 2 20 — — — 10 3 20 — — — 10 4 20 3.5 — — 10 5 203.5 — — 10 6 20 3.5 — — 10 7 — — 10 — 10 8 — — 10 — 10 9 — — 10 — 10 10— — 10 4.4 10 11 — — 10 4.4 10 12 — — 10 4.4 10

-   -   11. Incubate 30 minutes on ice.    -   12. Dilute 10×FACS Lysing Solution 1:10 with dH₂O. Add 1 ml of        FACS sing Solution to each tube. Vortex and incubate RT 15        minutes.    -   13. Spin the tubes for 5 minutes at 1500 rpm, at 4° C.    -   14. Resuspend in 200 μl FACS Lysing Solution.    -   15. Read on flow cytometer, adjusting compensation settings as        necessary. For acquisition, gate on the monocyte population, as        identified by forward and side scatter properties (G1). Gate on        CD14+ monocytes using a dot plot of forward scatter vs. CD14        (G2). The additive events of G1 and G2 (termed G3) are observed        on a histogram looking at the FL2 channel (CD86 PE, FUN1) or FL4        channel (CD86 APC, 2D4). Acquisition is stopped after        accumulation of 3000 G3 events.    -   16. For analysis, the median fluorescence of G3 events is        determined on a histogram of FL2 events (CD86 PE, HA5) or FL4        events (CD86 APC, 2D4) and is used to determine the relative        level of CD86 present on the surface of CD14+ monocytes.

Example 3 Clinical Assay

A. CD86 Clinical Assay Procedure

To perform such an assay, blood samples obtained from patients dosedwith L104EA29YIg can be aliquoted into 200 μl samples into a 12×75 mmpolystyrene tube on ice (4° C.). To each tube, 25 μg of mouse mixed IgGscan be added to block potential FcR binding of detection antibodies(e.g. FUN-1, 2D4, 1420 etc.) and incubated for 15 minutes at 4C. To eachtube, 20 μl anti-human CD14 FITC can be added and indicated amount (e.g.1 μg) of fluorescently labeled anti-human CD86 (e.g. FUN1, HA5 or 2D4)and incubated at 4° C. for 30 minutes. To assess non-specificfluorescence associated with the labeled anti-CD86 mAbs, excessunlabeled anti-human CD86 mAb is added to a subset of the relevantsamples (e.g. unlabeled HA5 is added to samples containing labeled HA5).2 ml of FACS Lysing Solution (BD) can be added to each tube to lyse thered blood cells and fix the leukocytes. The blood may be subsequentlyincubated at 4° C. for 30 minutes. To isolate the leukocytes and removelysed RBCs and excess reagents, the samples can be centrifuged at 1500rpm for 5 minutes at 4° C. and resuspended in 200 μl FACS LysingSolution for analysis on the flow cytometer as described in the methods.The total CD86 expressed on the surface of the APC can be determined byusing mAb 2D4 while the available CD86 can be measured by using mAbFUN-1 or HA5 as examples. Specific binding (ΔMFI) is determined by thedifference between the total binding (labeled anti-CD86 mAb alone) andthe non-specific binding (labeled +excess unlabeled anti-CD86 mAb). Withthis data one could calculate the ratio of unbound CD86 to total CD86and determine the extent of receptor saturation by L104EA29YIg in agiven blood sample. Alternatively, one could also perform the sameprocedure on patient blood obtained prior to administration ofL104EA29YIg to determine the total CD86 levels and repeat the analysisfollowing administration of the compound to measure the decrease in thebinding of the competing antibody as compared to pre-treatment. In thisway, one would be able to determine the extent of receptor saturationfollowing administration of compound.

B. CD80 Clinical Assay Procedure

Although CD80 expression on monocytes is very low or not at all, othercell types or activated monocytes do express higher levels of CD80. Theexpression of total and available CD80 can be measured in a similarfashion on these cell types by using the non competing CD80 mAb 1420 tomeasure total CD80 expressed and the competing mAb L307.4 or mAb MAB104to measure CD80 unbound by L104EA29YIg. With this data one couldcalculate the ratio of unbound CD80 to total CD80 and determine theextent of receptor saturation by L104EA29YIg in a given blood sample.Alternatively, one could also perform the same procedure on patientblood obtained prior to administration of L104EA29YIg to determine thetotal CD80 levels and repeat the analysis following administration ofthe compound to measure the decrease in the binding of the competingantibody as compared to pre-treatment. In this way, one would be able todetermine the extent of receptor saturation following administration ofcompound.

As shown in FIG. 20, whole blood was stimulated with 1 μg/ml LPS for 4hours at 37° C. to induce the expression of CD80. The unbound CD80 levelwas determined by the level of binding of competing antibody L307.4. Theresults shown in FIG. 20 represent the average response of 6 donors.

C. Allo-response Assay

To measure allo-responses, mixed leukocyte reactions (MLR) wereperformed. For MLR proliferation assays measuring titrations of CTLA-4Igor L104EA29YIg, T cells were cultured at 1×10⁵/well in quadruplicatewells together with 1×10⁴, 2×10³ or 0.4×10³ allogeneic MoDC asantigen-presenting cells (APCs) in 96-well round-bottom plates in atotal volume of 200 μl of 10% FCS-RPMI. CTLA-4Ig or L104EA29YIg wasadded to wells at a starting final concentration of 30 μg/ml, followedby half-log dilutions down to a final concentration of 1 ng/ml. On day 5after initiation of the MLR, cultures were pulsed with one μCi of³[H]-thymidine (PerkinElmer, Boston, Mass.) for 6 hours, harvested on aPackard cell harvester (PerkinElmer), and counted by liquidscintillation on a +Packard TopCount NXT (PerkinElmer). The results ofCD80 and CD86 receptor saturation and inhibition of allo-response assayare shown in FIG. 21.

Example 4 Methods of Producing Antibodies

The production of antibodies is discussed in detail in U.S. patentapplication Ser. No. 10/375157 (Publication No. 2003-0224458) which ishereby incorporated by reference in its entirety. The antibodies of theinvention can be produced by any method known in the art for thesynthesis of antibodies, in particular, by chemical synthesis orpreferably, by recombinant expression techniques.

The mAb 2D4 and mAB 1420 were labeled with fluorochromes typically usedin flow cytometry studies (FITC, PE, etc.). The assay is not dependenton the type of fluorochrome used, different labels can be used in thisassay and mixed and matched to suit the investigators purpose.

Throughout this application various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this invention pertains.

1. An assay method for monitoring binding efficiency of L104EA29YIg to areceptor, said method comprising the steps of: isolating peripheralmononuclear cells from a blood sample to provide test and controlmonocytes; treating the test monocytes with a predetermined amount ofL104EA29YIg; adding anti-receptor antibodies to control monocytes andtreated test monocytes; measuring binding of anti-receptor antibody insaid treated test and control monocytes;and determining the bindingefficiency of L104EA29YIg by comparing binding of anti-receptor antibodyto said control monocytes relative to said treated test monocytes. 2.The assay of claim 1, wherein the receptor is CD86.
 3. The assay ofclaim 2, wherein the anti-receptor antibody is anti-CD86 mAb.
 4. Theassay of claim 3, wherein the anti-receptor antibody is FUN-1.
 5. Theassay of claim 3, wherein the anti-receptor antibody is IT2.2.
 6. Theassay of claim 3, wherein the anti-receptor antibody is HA5.2B7.
 7. Theassay of claim 1, wherein the receptor is CD80.
 8. The assay of claim 7,wherein the anti-receptor antibody is mAb L307.4
 9. The assay of claim7, wherein the anti-receptor antibody is mAb MAB
 104. 10. An assaymethod for monitoring binding efficiency of L104EA29YIg to a receptor,said method comprising the steps of: isolating peripheral mononuclearcells from a blood sample; adding L104EA29YIg to said blood sample;adding competing anti-receptor antibodies to said blood sample; addingnon-competing anti-receptor antibodies to said blood sample; measuringthe binding of competing anti-receptor antibody; measuring the bindingof non-competing anti-receptor antibody; and comparing the binding ofthe non-competing anti-receptor antibody (total receptor binding) to thebinding of the competing anti-receptor antibody to determine the bindingefficiency of L104EA29YIg.
 11. The assay method of claim 10, wherein thereceptor is CD86.
 12. The assay method of claim 11, wherein thecompeting anti-receptor antibody is anti-CD86 mAb.
 13. The assay methodof claim 12, wherein the competing anti-receptor antibody is FUN-1 14.The assay method of claim 12, wherein the competing anti-receptorantibody is IT2.2.
 15. The assay method of claim 12, wherein thecompeting anti-receptor antibody is HA5.2B7.
 16. The assay method ofclaim 11, wherein the non-competing anti-receptor antibody is 2D4. 17.The assay method of claim 10, wherein the receptor is CD80.
 18. Theassay method of claim 17, wherein the competing anti-receptor antibodyis mAb L307.4.
 19. The assay method of claim 17, wherein the competinganti-receptor antibody is mAb MAB
 104. 20. The assay method of claim 17,wherein the non-competing receptor antibody is mAb
 1420. 21. An antibodymAb 2D4, produced by hybridoma cell line Mus muscalis, spleen cell: 2D4,deposited under ATCC Deposit No. PTA-7305.
 22. A hybridoma cell line Musmuscalis, spleen cell: 2D4, deposited under ATCC Deposit No. PTA-7305,producing the antibody mAb 2D4.
 23. An antibody 1420, produced byhybridoma cell line Mus muscalis, spleen cell: 1420 deposited under ATCCDeposit No. PAT-7304.
 24. A hybridoma cell line Mus muscalis, spleencell 1420 deposited under ATCC Deposit No. PAT-7304, producing theantibody mAb
 1420. 25. An assay method for monitoring binding efficiencyof L104EA29YIg to a receptor in a patient, said method comprising thesteps of: (a) obtaining a blood sample from a patient dosed withL104EA29YIg, the blood sample including leucocyte cells; (b) adding alabeled marker to identify a subset of leukocyte cells of interest; (c)adding labeled competing and non-competing anti-receptor antibodies tothe blood sample; (d) lysing the blood sample and fixing leukocytes; (e)isolating leukocytes from the lysed blood sample; (f) measuring thetotal receptor expression and available receptor from said subset ofleukocyte cells of interest.
 26. The assay of claim 25, wherein thereceptor is CD86.
 27. The assay of claim 26, wherein the competinganti-receptor antibody is mAb FUN-1.
 28. The assay of claim 26, whereinthe competing anti-receptor antibody is mAb IT2.2.
 29. The assay ofclaim 26, wherein the competing anti-receptor antibody is mAB HA5.2B7,30. The assay of claim 26, wherein the non-competing anti-receptorantibody is mAb 2D4.
 31. The assay of claim 25, wherein the receptor isCD80.
 32. The assay of claim 31, wherein the competing anti-receptorantibody is mAb L307.4.
 33. The assay of claim 31, wherein the competinganti-receptor antibody is mAb MAB104.
 34. The assay of claim 31, whereinthe non-competing anti-receptor antibody is mAb
 1420. 35. The assay ofclaim 1, further comprising detecting the presence of antibodies usingflow cytometry.
 36. The assay of claim 10, further comprising detectingthe presence of antibodies using flow cytometry.
 37. The assay of claim25, further comprising detecting the presence of antibodies using flowcytometry.
 38. The assay of claim 25, wherein subsequent to step (a) andprior to step (b), further comprising step (a′) blocking Fcreceptor-mediated non-specific binding.
 39. The assay of claim 38,wherein the Fc receptor-mediated non-specific binding is blocked by theaddition of mixed mouse IgGs to the blood sample.
 40. The assay of claim25, wherein the marker added to identify a subset of monocyte cells isCD14-FITC.
 41. The assay of claim 25, wherein the subset of leukocytecells of interest are monocytes or dendritic cells.
 42. The assay ofclaim 25, wherein the binding efficiency of L104EA29YIg is measured bycomparing bound and unbound receptors to determine percent of receptorsaturation by L104EA29YIg.
 43. An assay method for monitoring bindingefficiency of L104EA29YIg to a receptor in a patient, said methodcomprising the steps of: (a) obtaining a control blood sample from saidpatient; (b) dosing said patient with L104EA29YIg, (c) obtaining a testblood sample from said patient dosed with L104EA29YIg; (d) adding alabeled marker to identify a subset of leukocyte cells of interest; (e)adding labeled competing anti-receptor antibody to the control and testblood samples; (f) lysing the blood samples and fixing leukocytes; (g)isolating leukocytes from the lysed blood sample; (h) comparing thebinding of the competing anti-receptor antibody in test and controlblood samples.
 44. The assay of claim 43, wherein the receptor is CD86.45. The assay of claim 44, wherein the competing anti-receptor antibodyis mAb FUN-1.
 46. The assay of claim 44, wherein the competinganti-receptor antibody is mAb IT2.2.
 47. The assay of claim 44, whereinthe competing anti-receptor antibody is mAb HA5.2B7.
 48. The assay ofclaim 43, wherein the receptor is CD80.
 49. The assay of claim 48,wherein the competing anti-receptor antibody is mAb L307.4.
 50. Theassay of claim 48, wherein the competing anti-receptor antibody is mAbMAB104.
 51. The assay of claim 42, further comprising detecting thepresence of antibodies using flow cytometry.